Team:Bielefeld-Germany/Protocols/Materials

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Materials: These enzymes, kits and materials were used in our project.

Used enzymes

Enzyme	Producer
AgeI	[http://www.fermentas.de/product_info.php?info=p247 Fermentas]
DpnI	[http://www.fermentas.de/product_info.php?info=p296 Fermentas]
EcoRI	[http://www.fermentas.de/product_info.php?info=p336 Fermentas]
GoTaq DNA-polymerase	[http://www.promega.com/products/pcr/routine-pcr/gotaq-pcr-core-systems/ Promega]
KOD Hotstart DNA-polymerase	[http://www.merck-chemicals.com/germany/life-science-research/kod-hot-start-dna-polymerase/EMD_BIO-71086/p_iFCb.s1O874AAAEj2Bl9.zLX Novagen]
NgoMIV	[http://www.neb.com/nebecomm/products/productR0564.asp NEB]
OneTaq DNA-polymerase	[http://www.neb.com/nebecomm/products/productM0480.asp NEB]
Pfu DNA-polymerase	[http://www.promega.com/products/pcr/routine-pcr/pfu-dna-polymerase/ Promega]
PstI	[http://www.fermentas.de/product_info.php?info=p458 Fermentas]
Phusion HF DNA-polymerase	[http://www.finnzymes.com/pcr/phusion_high_fidelity_dna_polymerase.html Finnzymes]
Shrimp alcaline phosphatase	[http://www.fermentas.de/product_info.php?info=p592 Fermentas]
SpeI	[http://www.fermentas.de/product_info.php?info=p202 Fermentas]
T4-DNA-Ligase	[http://www.fermentas.de/product_info.php?info=p580 Fermentas]
T5 exonuclease	[http://www.neb.com/nebecomm/products/productM0363.asp NEB]
taq DNA Ligase	[http://www.neb.com/nebecomm/products/productM0208.asp NEB]
taq DNA-polymerase	[http://www.bioline.com/h_prod_detail.asp?itemid=219 Bioline]

Used Kits

Function	Name
Molecular Cloning	[http://www.fermentas.de/product_info.php?info=p1620 Fermentas CloneJET™ PCR Cloning Kit]
Plasmid purification	[http://www.fermentas.de/product_info.php?info=p874 Fermentas GeneJET™ Plasmid Miniprep Kit]
Plasmid purification	[http://www.promega.com/b/de/minis/default.aspx?utm_source=Promega&utm_medium=Online&utm_campaign=Online_DEPureYield100Preps_Promega_HP_Banner Promega PureYield™ Plasmid Preps]
PCR Cleanup	[http://www.mn-net.com/tabid/10745/default.aspx Macherey Nagel NucleoSpin® Extract II]
PCR Cleanup	[http://www.promega.com/products/dna-and-rna-purification/dna-fragment-purification/wizard-sv-gel-and-pcr-clean_up-system/ Promega Wizard® SV Gel and PCR Clean-Up]
PCR core system	[http://www.promega.com/products/pcr/routine-pcr/gotaq-pcr-core-systems/ Promega GoTaq® PCR Core System I]

Media, buffer, solutions etc.

Ampicillin stock solution

Solubilize 100 mg mL^-1 Ampicillin
Store at -20 °C

Chloramphenicol stock solution

Solubilize 20 mg mL^-1 Chloramphenicol
Store at -20 °C

TAE buffer

For 1 L of 50 x TAE buffer you need:

242.48 g Tris
41.02 g Sodiumacetate
18.612 g EDTA
Adjust pH to 7.8 with acetic acid
Solve in dH₂O

10 mL of the stock is diluted in 1 L dH₂O for the gel electrophoresis (0.5 x TAE buffer).

DNA loading buffer

50 % (v/v) glycerol
1 mM EDTA
0.1 % (w/v) bromphenol blue
Solve in ddH₂O

LB medium

For 1 L of LB medium you need:

10 g Trypton
5 g yeast extract
10 g NaCl
12 g Agar-Agar (for plates)
Adjust pH to 7.4

Autoinduction medium for KRX

This medium is based on the LB medium.

After heat sterilization of 900 mL add the following chemicals

5 mL of a 200 g L^-1 steril L-rhamnose stock solution (final concentration 2 g L^-1 L-rhamnose)
2.5 mL of a 200 g L^-1 steril glucose stock solution (final concentration 0.5 g L^-1 glucose)
if necessary add antibiotics
- Cm: 1 mL of a 20 μg mL^-1 Cm stock solution (final concentration 2 μg L^-1)
- Amp: 1 mL of a 100 μg mL^-1 Amp stock solution (final concentration 100 μg L^-1)
fill-up to 1 L with steril ddH₂O

M9 medium

For 250 mL M9 medium you need 175 mL sterile water (for plates add 4 g Agar-Agar as well). Then add (in the following order:

250 µL 100 mM CaCl₂
2.5 mL trace salts
- store this stock solution in the dark
250 µL MgSO₄
250 µL 50 mM FeCl₃ / 100 mM citrate (one solution, citrate is iron carrier)
- store this stock solution cold and in the dark
carbon source stock solution (e.g. glucose)
50 mL 5x M9 salts stock solution
- 64 g L^-1 Na₂HPO₄ * 7 H₂O
- 15 g L^-1 KH₂PO₄
- 2.5 g L^-1 NaCl
- 5 g L^-1 NH₄Cl
antibiotic stock solution
fill up to 250 mL with sterile water

HSG medium

14.9 g L^-1 glycerine
13.5 g L^-1 soy peptone
7 g L^-1 yeast extract
2.5 g L^-1 NaCl
2.3 g L^-1 K₂HPO₄
1.5 g L^-1 KH₂PO₄
0.249 g L^-1 MgSO₄ * 7 H₂O

5x isothermal reaction buffer for Gibson assembly

storage -20˚C

3 mL of 1 M Tris-HCl (pH 7.5)
150 µL of 2 M MgCl2,
60 µL of 100 mM dGTP
60 µL of 100 mM dATP
60 µL of 100 mM dTTP,
60 µL of 100 mM dCTP
300 µL of 1 M DTT
1.5 g PEG-8000 and
300 µL of 100 mM NAD

Cold osmotic shock buffers for the release of periplasmic protein fraction

Cell fractionating buffer #1 (pH 8)

0.2 M Tris
200 g L ^-1 sucrose
0.1 M EDTA

Cell fractionating buffer #2 (pH 8)

0.01 M Tris
0.005 M MgSO₄

Denaturation buffer for inclusion bodies

6 M urea
50 mM Tris-HCl
10 mM MgCl₂

Buffers for S-layer IEX

Binding Buffer:

25 mM Sodiumacetate, pH 6
25 mM NaCl

Elution Buffer:

25 mM Sodiumacetate, pH 6
1 M NaCl

SDS-PAGE gel

The following amouts are for one gel. Stacking gel 5 %:

775 μL H₂O
1.25 mL 0,25 M Tris (pH 6,8)
425 μL Bis/Acrylamide (0,8 %, 30 %)
50 μL 5 % SDS
25 μL 10 % Ammonium persulfate
3 μL TEMED

Separating gel 12 %:

1.5 mL H₂O
2.8 mL 1 M Tris (pH 8,8)
3.0 mL Bis/ Acrylamide (0,8%, 30%)
150 μL 5% SDS
37.5 μL 10% Ammonium persulfate
5 μL TEMED

SDS running buffer

25 mM Tris [pH 8,3]
192 mM Glycerol
0.1 % SDS

4x Laemmli-buffer

250 mM Tris-HCl
40 % [v/v] Glycerol
20 % [v/v] 2-Mercapthoethanol
80 g L^-1 SDS
0,04 g L^-1 BPB

Colloidal Coomassie Brilliant Blue G-250 staining solution

for 1 L staining solution

dissolve 50 g L^-1 (NH4)₂SO₄ in ddH₂O
add 10 % (v/v) ethanol
dissolve 0,2 g L^-1 Coomassie Brilliant Blue G-250
add 2 % (v/v) phosphoric acid
fill up to 1 L with ddH₂O

Silver staining solutions

Fixation solution:

50 % (v/v) ethanol
12 % (v/v) acetic acid
1 mL L^-1 formaldehyde (37 %)

Thiosulfate solution:

0.1 g L^-1 Na₂S₂O₃

Impregnation solution:

2 g L^-1 silver nitrate (AgNO₃)
0.75 mL L^-1 formaldehyde (37 %)

Developing solution:

120 g L^-1 sodium carbonate (Na₂CO₃)
1 mL L^-1 formaldehyde (37 %)

Stop solution:

18.6 g L^-1 EDTA

Binding buffer

20 mM Na₃PO₄, pH 7,4
500 mM NaCl
20 mM Imidazole

NPI-10 (lysis buffer)

50 mM NaH₂PO₄, pH 8,0
300 mM NaCl
10 mM Imidazole
2 mM PMSF

NPI-20 (wash buffer)

50 mM NaH₂PO₄, pH 8,0
300 mM NaCl
20 mM Imidazole

NPI-500 (elution buffer)

50 mM NaH₂PO₄, pH 8,0
300 mM NaCl
500 mM Imidazole

Deadenylation buffer

20 mM Tris-HCl, pH 7.5
50 mM NaCl
4 mM MgCl₂
1 mM EDTA
1 mM DTT
1 mM NMN

DNA ligase buffer

20 mM Tris-HCl, pH 7.5
50 mM NaCl
20 % [v/v] Glycerol

NAD⁺ bioassay buffer

50 mM Tris-HCl, pH 8,0
10 mM MgCl₂
2.5 mM CaCl₂
5 mM DTT
0.05 % BSA

Buffers for His-Tag affinity chromatography

For denaturing conditions add 6 M Urea
Adjust pH to 7.4 - 7.6

Buffer	Sodium phosphate [mM]	NaCl [mM]	Imidazole [mM]
Binding buffer	20	500	5
Elution buffer 1	20	500	40
Elution buffer 2	20	500	60
Elution buffer 3	20	500	100
Elution buffer 4	20	500	300
Elution buffer 5	20	500	500

Used chemicals

Chemical	Producer	Purity
Acetonitrile	VWR	99.9 %, HPLC Grade
Bisphenol A	[http://www.sigmaaldrich.com/catalog/Lookup.do?N5=All&N3=mode+matchpartialmax&N4=bisphenol+a&D7=0&D10=bisphenol+a&N1=S_ID&ST=RS&N25=0&F=PR Sigma]	97 %
Bisphenol F	[http://www.alfa.com/de/GP100W.pgm?DSSTK=A11417&rnd=097380668 Alfa Aesar]	98 %
Ethylacetate	VWR	> 99.5 %, p.a.
Isopropyl β-D-1-thiogalactopyranoside	[http://www.carlroth.com/catalogue/catalogue.do;jsessionid=D5D58FA281A3E75342D9198F07546935?id=17413&favOid=000000010000911300020023&act=showBookmark&lang=de-de&market=DE Roth]	≥ 99 %
L-rhamnose	[http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=de&N4=83650\|FLUKA&N5=SEARCH_CONCAT_PNO\|BRAND_KEY&F=SPEC Fluka]	≥ 99 %
Chemical A	[http://www.link.com Producer A]	XX.X %